1. Field of the Invention
The present invention relates generally to a vaccine composition and methods of preventing and treating infection in humans and animals therewith. More specifically, the invention relates to a mutant rabies virus wherein the nucleoprotein is mutated at the amino acid wherein phosphorylation occurs. The invention also relates to vectors for delivering a gene to a human or animal, and methods of delivering the gene thereto.
2. Description of the Related Art
Within the Rhabdoviridae family, rabies virus is the prototype of the Lyssavirus genus and vesicular stomatitis virus (VSV) is the prototype of the Vesiculovirus genus (Wagner and Rose, 1996). The genomic RNA is encapsidated with nucleoprotein (N) and this N-RNA complex, together with the phosphoprotein (P, also termed as NS) and RNA-dependent RNA polymerase (L), forms the RNP complex. The N protein of the rhabdoviruses, like the N protein from other members in the order of the mononegavirales, plays vital roles in regulating viral RNA transcription and replication by encapsidating de novo synthesized viral genomic RNA. Although rabies virus N and VSV N do not share a high degree of homology in the primary nucleotide and protein sequences, they do have conserved regions and similar protein characteristics. For example, the N protein of rabies virus has four conserved amino acid stretches homologous with those of VSV (Tordo et al., 1986). In addition, a similar helical structure of N protein exists in both rabies virus and VSV, with an xcex1-helix continuing from the N-terminus through most of the protein, and a xcex2-turn towards the C-terminus (Barr et al., 1991).
One major structural difference, however, exists between rabies virus N and VSV N. Rabies virus N is phosphorylated while VSV N is not (Sokol and Clark, 1973). The phosphorylation has been mapped to serine residue at position 389 of the rabies virus N (Dietzschold et al., 1987). Previously, it was demonstrated that dephosphorylation of rabies virus N or mutation of the serine 389 to alanine resulted in increased binding to in vitro-synthesized leader RNA (Yang et al., 1999). Furthermore, mutation of the phosphorylated serine to alanine resulted in reduction of viral transcription and replication of a rabies virus minigenomic RNA (Yang et al., 1999). However, in the minigenome system, viral proteins necessary for viral transcription and replication were synthesized by T7 polymerase, and thus their synthesis was not under the control of rabies virus regulatory machinery.
Rabies has always had an aura of tragedy and mystery. Its dramatic clinical expression and almost always fatal outcome guarantee that rabies prevention is given high priority. Despite significant progress in biological research, rabies remains a significant global disease. Annually, more than 70,000 human fatalities are estimated, and millions of others require post-exposure treatment (Meslin et al., 1994; Anonymous, 1993). Although humans are the dead-end host, the disease is epizootic or enzootic in domestic animals as well as in wildlife (Fu, 1997; Rupprecht et al., 1995; Smith et al., 1995). Dogs remain the most important reservoir in Asia, Africa, and Latin America where most human rabies cases occur (Fu, 1997). In countries where dog rabies is controlled through animal vaccination, the number of human cases has been reduced considerably (Smith et al., 1995). However, rabies in wildlife presents a more challenging problem in these countries (Rupprecht et al., 1995; Smith et al., 1995). Fox rabies has been endemic in Europe and North America for many years, although a recent endeavor in oral vaccination has been successful in reducing or even eliminating rabies in many parts of Europe (Brochier et al., 1991). In the United Sates, wildlife rabies accounted for more than 90% of the reported rabies cases (more than 7,000 each year) in the past decade (Rupprecht et al., 1995; Smith et al., 1995; Krebs et al., 2000a; Krebs et al., 2000b) and there are at least five major wildlife rabies reservoirs that maintain concurrent epizootics (Smith et al., 1995). Epizootic raccoon rabies continues to occur in all the states along the eastern seaboard, and it is spreading westwards to Ohio, West Virginia, and Alabama (Krebs et al., 2000b). Skunk rabies remains enzootic in the central states and California (Krebs et al., 2000b). Fox rabies occurs sporadically in Arizona, Alaska, and Texas and also in the eastern states where raccoon rabies is epizootic (Krebs et al., 2000b). Bat rabies is widely distributed throughout the 48 contiguous states (Krebs et al., 2000b). These epizootics of wildlife rabies present a health threat for humans. Therefore, controlling rabies and protecting humans from rabies virus infection requires multi-layered control strategies, particularly vaccination of humans before or after exposure, regular vaccination of pet animals, and vaccination of wildlife.
Vaccination of humans after exposure can be dated back to the time of Pasteur when he injected Joseph Meister with attenuated rabies virus made from neuronal tissue (Pasteur et al., 1996). Since then, human rabies vaccines have gone through successive improvements, particularly the development of human diploid cell culture vaccine (HDCV) by Koprowski and associates at the Wistar Institute (Wiktor et al., 1964). The tissue culture vaccine is not only safe compared with the old brain vaccines because it does not contain neuronal tissues, but it is also more effective. People immunized with HDCV developed high virus neutralizing antibody (VNA) titers as early as 10 days after inoculation, compared with those immunized with the nervous tissue vaccine in whom neutralizing antibody titers do not reach protective levels until 30 days after the immunization (Wiktor et al., 1964). Today, many of the derivatives of tissue culture vaccines are similar to HDCV, and they are both effective and well tolerated. They include the purified chicken embryo cell vaccine (PCEC, Barth et al., 1984; Sehgal et al., 1993), the purified Vero cell rabies vaccine (PVRV, Suntharasamai et al., 1986) and the purified duck embryo cell vaccine (PDRV, Khawplod et al., 1995). A typical post-exposure treatment for an individual bitten by a rabid or a suspected rabid animal consists of the prompt administration of multiple injections of one of the above-mentioned tissue culture vaccines. Depending upon the nature and severity of the bite, it is also recommended that individuals receive antirabies antiserum prepared either in animals (usually equine) or preferably in humans (human rabies immune globulins, or HRIG) (Anonymous, 2000). Less frequently and under special circumstances, humans considered at risk of inapparent rabies exposure, such as animal control officers, veterinarians, and laboratory personnel working with the virus, are immunized against rabies, which is known as pre-exposure vaccination (Anonymous, 2000).
Although the tissue culture vaccines are safe and effective, there are problems. Because all these vaccines are made from inactivated viruses, multiple doses over an extended time period are required to stimulate optimal immune responses (Anonymous, 2000). Failure to complete the whole series of vaccination may result in the development of diseases (Shill et al., 1987; Lumbiganon et al., 1987; Anonymous, 1988). Allergic reactions to proteins contained within cell culture vaccines occur in approximately 6% of the vaccinees given booster injections (CDC, 1984). Indeed, there is some evidence that the most serious adverse reactions are to human albumin denatured by the xcex2-propiolactone used to inactivate the virus (Warrington et al., 1987; Swanson et al., 1987; Anderson et al., 1987). Furthermore, the high cost of these tissue culture vaccines makes it difficult to effectively utilize in developing countries where it is needed most. Post-exposure treatment may exceed $2,000 to 3,000 dollars (in the United States) per case (Melter, 1996). Most human rabies cases occur in developing countries, where vaccinees cannot afford to pay this amount. Thus, a frequently used vaccine for rabies in developing countries is from animal neural tissue, usually produced either in livestock or in suckling mouse brains (Fuenzalida vaccine) (Fuenzalida, 1972). Twenty-one doses of the nervous tissue vaccine are usually required by intraperitoneal injection, and such vaccines may cause neurological diseases (Trejos et al., 1974).
Vaccination of pets (dogs and cats) in the United States is carried out as recommended in the Compendium of Animal Rabies Prevention and Control by the National Association of State Public Health Veterinarians (Anonymous, 2000). Usually pet animals are immunized at 6 weeks of age and revaccinated annually or triennially depending on the vaccines used (Anonymous, 2000). Most of the licensed rabies vaccines for pets are inactivated rabies viruses. Recently, a recombinant canary pox virus expressing rabies virus G was approved for cat immunization (Anonymous, 2000). Although these vaccines provide adequate protection in dogs ant cats, the vaccines do induce local reactions (Wilcock et al., 1986). Furthermore, multiple immunizations are required to maintain sufficient immunity throughout life (Anonymous, 2000). Dogs immunized repeatedly with commercial vaccines may not always maintain adequate titers, and only one-third of the dogs showed VNA titers above the 1:5 base line (Tims et al., 2000). In addition, vaccination of puppies less than 3 months of age fails to induce protective immunity, although the maternal antibodies transferred from bitches declined to undetectable levels by 6 weeks of age (Aghomo et al., 1990). There is a period from the time of the waning of maternal antibody to the time of active immunity in which the young animals may not be protected (Mitmoonpitak et al., 1998; Clark et al., 1996).
Wildlife rabies exists in many countries and continues to present a major public health threat. Efforts to control wildlife rabies during the past two decades in both Europe and North America have been directed towards oral vaccination (Baer, 1988). Initially, an attenuated rabies virus, Street Alabama Dufferin B19 (SAD) strain was used, and it did not cause rabies when orally administered to foxes (Baer, 1988). Field trials to vaccinate red foxes in European countries with SAD in chicken-head baits resulted in more than 60% of rabies immune foxes and stopped the spread of the disease into untreated areas (Wandeler et al., 1998; Schneider et al., 1988). However, SAD still causes disease in rodents (Winkler et al., 1976) and in domestic animals (Esh et al., 1982). Subsequently, a recombinant vaccinia virus expressing the rabies virus G (VRG) was developed (Kieny et al., 1984) and was found to be an effective oral immunogen for raccoons and foxes under laboratory conditions (Rupprecht et al., 1986; Blancou et al., 1986). Further testing of VRG in fishmeal baits was carried out in animals in the wild, and it has been demonstrated that VRG is safe (Brochier et al., 1989; Rupprecht et al., 1993). Neither vaccine-associated morbidity or mortality nor gross lesions or detrimental side effects have been associated with vaccination in target and non-target animal species. The vaccine is also efficacious in inducing protective immunity and field application with VRG resulted in large-scale elimination of fox rabies in vaccinated areas in Europe (Brochier et al., 1991). Similar application of VRG in the United States has resulted in a blockade of coyote rabies spreading in Texas (Fearneyhough et al., 1998), and raccoon rabies spreading in other states (Hanlon et al., 1998; Robbins et al., 1998; Roscoe et al., 1998). Although VRG is safe in vaccinated animals and efficacious in stimulating immunity in target animal species, a recent incident involving a pregnant woman underscores the risks of using such recombinant vaccines even in wildlife animals, particularly in densely populated areas (Rupprecht et al., 2001). The woman was bitten on the finger and left forearm when she tried to remove a VRG recombinant virus-laden bait from her dog""s mouth. Within 10 days she developed an intensive local inflammatory reaction around two necrotic lesions at the forearm bite sites and adenitis. She went on to develop generalized erythroderma that eventually subsided after exfoliation (Rupprecht et al., 2001). This incident casts doubts on the future use of VRG as a rabies vaccine for wildlife.
It is clear that current vaccines used in humans and other animals have problems in safety, effectiveness, and cost. More effective, safe, and inexpensive vaccines are needed for controlling rabies in animals and prevent human rabies. Many novel vaccines are being developed and tested including DNA vaccines and other recombinant vaccines. DNA vectors expressing rabies virus G have been found to stimulate both T helper cells and the production of rabies virus VNA (Xiang et al., 1994). Furthermore, immunization of mice with these DNA vectors protected mice and monkeys against subsequent challenge infections with lethal rabies virus (Xiang et al., 1994; Ray et al., 1997; Lodmell et al., 1998). However, induction of immune responses by DNA vaccines usually takes longer, and the magnitude of the immune response is lower compared with conventional vaccines (Xiang et al., 1994; Osorio et al., 1999). Recombinant human adenoviruses expressing rabies virus G have also been developed (Prevec et al., 1990; Xiang et al., 1996). The recombinant adenovirus vaccines can induce VNA and protect vaccinated animals (mice, dogs, skunks, and foxes) against challenge infection (Tims et al., 2000; Prevec et al., 1990; Xiang et al., 1996; Charlton et al., 1992; Wang et al., 1997). There is concern with the adenoviral vector in the immune responses directed to the adenoviral proteins (Wang et al., 1997). Preexisting anti-adenoviral immunity may prevent the uptake of the vaccine by cells needed for expression of the target gene and, thus, impair the active immune response to the target antigen. Furthermore, revaccination or vaccination with adenoviral vector expressing a different target antigen may no longer be effective.
Live-attenuated virus vaccines have long been known to be more effective in inducing long-lasting humoral and cell-mediated immunity, and many diseases are controlled or eradicated by using live modified viral vaccines. The global eradication of small pox is essentially achieved by using a less virulent cowpox virus vaccine (Henderson, 1980). Poliomyelitis is on the verge of global eradication because of the mass vaccination with the live polio vaccines (Sabin et al., 1973). Many other viral diseases such as measles, mumps, and rubella, just to name a few, are brought under control by live modified virus vaccines (Arvin, 2000). Thus, a live modified rabies virus vaccine may have the advantage over currently licensed vaccines by providing long-lasting immunity and reducing the doses required. As a result, the cost will be lowered markedly. However, such live modified rabies vaccines must be completely avirulent, particularly for humans. To this end, the SAD strain of rabies virus, which was initially used for wildlife vaccination in the 1980s (Baer, 1988; Wandeler et al., 1998; Schneider et al., 1988), was further attenuated by successive selection using neutralizing monoclonal antibodies (Mab), resulting in the selection of strains SAG1 and SAG2 (Le Blois et al., 1990; Flamand et al., 1993; Schumacher et al., 1993; Lafay et al., 1994). The selection of SAG1 and SAG2 using Mabs was based on earlier findings that mutation of the glycoprotein at arginine 333 reduced the virulence of the rabies virus (Dietzschold et al., 1983; Seif et al., 1985). The SAG1 virus possesses one mutation at position 333, where arginine is replaced by lysine (Lafay et al., 1994). Compared with the SAD strain, which is still pathogenic in adult mice by intracerebral (i.c.) route of inoculation, the SAG1 virus is avirulent when given to adult mice by i.e., intramuscular (i.m.), and per os (Le Blois et al., 1990; Lafay et al., 1994). The SAG1 virus is as effective as SAD in vaccinating foxes via the oral route (Le Blois et al., 1990). To stabilize the avirulent virus, a SAG2 virus was selected with an additional Mab (Lafay et al., 1994). SAG2 bears double mutations at position 333, changing from arginine (AGA) to glutamic acid (GAA), which reduces further the possibility of the virus to revert to virulent wild-type (wt) (Schumacher et al., 1993; Lafay et al., 1994). The SAG2 virus is avirulent for adult rodents, foxes, cats, and dogs by any route of inoculation (Schumacher et al., 1993; Lafay et al., 1994). Oral vaccination of foxes and dogs has resulted in the protection against a lethal challenge with rabies virus. Field trials with SAG2 in immunizing foxes and dogs demonstrated its safety and immunogenicity (Masson et al., 1996). However, SAG2 can induce rabies in suckling animals by i.c. inoculation (Schumacher et al., 1993; Lafay et al., 1994), raising the possibility that younger animals or immunocompromised animals may still be infected with the virus and develop disease.
These selected mutant viruses with changes on the arginine 333 of the G replicate well in cell culture, suggesting that the rate of viral replication of these viruses is not affected (Lafay et al., 1994). However, investigation of the ability of these viruses to invade the nervous system revealed that the virus can invade the first order neurons but fails to spread to secondary or tertiary neurons, indicating that the ability of these mutant viruses to spread in the nervous system via synaptic junctions is reduced (Coulon et al., 1989). Synaptic spreading is the major route for virus dissemination in the adult CNS (Gosztonyi et al., 1993). In the neonatal animals, synaptic spreading may not be the only way for virus dissemination. Because myelin development may not be complete in neonatal animals, the rabies virus may also spread from infected to uninfected neurons in neonatal animals by budding from infected neurons and infecting other uninfected neurons, more or less like that in cell cultures (Dietzschold et al., 1985). Therefore, reducing the rate of viral replication may be necessary to develop avirulent rabies virus vaccines. Like other single-stranded, non-segmented RNA viruses, rabies virus transcription and replication is regulated by the complicated interaction between the components within the ribonucleoprotein complex (RNP). RNP is composed of the genomic RNA, which is encapsidated by the nucleoprotein (N), together with the phosphoprotein (P) and the RNA-dependent RNA polymerase (L) (Wunner, 1991). Theoretically, mutation of these viral proteins may result in a reduced rate of replication for the rabies virus. With the recent development of reverse genetics technology for negative-stranded RNA viruses (Enami et al., 1990; Pattnaik et al., 1990; Pattnaik et al., 1991; Conzelmann et al., 1994; Schnell et al., 1994), manipulation of the viral genome for this group of viruses became possible (Conzelmann et al., 1994; Schnell et al., 1994; Lawson et al., 1995; Whelan et al., 1995). Application of this technology has resulted in a better understanding of how this group of viruses regulates their transcription and replication (Enami et al., 1990; Pattnaik et al., 1990; Pattnaik et al., 1991; Conzelmann et al., 1994; Schnell et al., 1994; Lawson et al., 1995; Whelan et al., 1995) and how each of the viral proteins functions in the replication cycle (Pattnaik et al., 1990) and in their pathogenicity (Etessami et al., 2000). Application of this technology has also resulted in attenuation of these viruses and some of them could be developed as vaccines (Wertz et al., 1998) or vectors for gene therapy (Finke et al., 1997).
N is the first product transcribed from the viral genome and is expressed abundantly in infected cells for all the negative-stranded RNA viruses (Wunner, 1991). N has been proposed to play a crucial role in the transition from RNA transcription to replication by encapsidating the nascent genomic RNA (Wunner, 1991). Mutation of the N could potentially lead to an attenuated phenotype. Indeed, moving the nucleoprotein (N) to other locations on the viral genome resulted in attenuation of vesicular stomatitis virus (VSV) (Wertz et al., 1998). This is caused by the inhibition of viral replication because of the reduced expression of the N protein. Recently, we have constructed mutant rabies virus with changes on the phosphorylation site of the N and found that the rate of viral replication was reduced by more than five-fold and the virus production was reduced by more than 10,000 times, indicating attenuation of the mutant rabies viruses (Wu et al., 2002).
Rabies still presents a public health threat causing more than 70,000 human deaths each year. Humans get infected with the rabies virus mostly through bites from rabid domestic and wildlife animals. Controlling rabies virus infection in domestic and wildlife animals, therefore, not only reduces the mortality in these animals but also reduces the risks of human exposure. Pre-exposure vaccinations for people who are constantly at risk further prevent human rabies, as do post-exposure immunizations for people who are bitten by rabid or suspected rabid animals. In the past few years, a recombinant vaccinia virus expressing rabies virus glycoprotein (VRG) has been used to control rabies in wildlife. Inactivated rabies virus vaccines are used to immunize domestic animals, particularly pets. Purified and inactivated rabies virus vaccines are used for humans in the pre- or post-exposure settings. Although these vaccines are effective, annual vaccinations are required to maintain adequate immunity in pets. For humans, multiple doses of the inactivated tissue culture vaccines are required to stimulate optimal immune responses. Furthermore, current tissue culture vaccines are expensive; thus most people in need of vaccinations (in developing countries) cannot afford them. Hence, there is a need to develop more efficacious and affordable rabies virus vaccines.
Therefore, in view of the aforementioned deficiencies attendant with prior art methods of vaccinating humans and animals against rabies virus, it should be apparent that there still exists a need in the art for a safe and cost-effective method therefor.
In accordance with the present invention, a mutant virus is provided which contains a mutation at a phosphorylation site in one or more of the proteins of the virus, which mutation causes the virus to be attenuated, and therefore, an improved vaccine composition can be produced therewith.
In particular, a mutant rabies virus is provided, wherein the virus contains a mutant rabies virus N protein which has an amino acid other than serine at position 389. Additionally, the mutant virus may contain one or more mutations within the N protein, or in other of the viral proteins, for example, in the G glycoprotein.
The invention also relates to vaccine compositions which contain the mutant virus, as well as to methods of inducing an immune response, and of protecting mammals from infection by rabies virus.
Also included in the invention are methods of producing the mutant virus and mutant viral proteins, including producing the mutant virus in a host cell which produces or even overproduces a wild-type counterpart of the mutant viral protein, which complements the other viral proteins such that production of the mutant viral particle is optimized. The invention also includes those host cells in which viral production is optimized.
Also included within the invention are nucleic acids which encode the mutant viral protein(s), and nucleic acids which encode a portion of, or the entire viral nucleic acid sequence. In addition, the invention includes vectors containing the nucleic acid sequences, including expression vectors, and host cells transformed with the nucleic acid sequences.
The invention also includes the viral proteins encoded by the mutant nucleic acids, vaccine compositions including the viral proteins, either alone or in combination with the intact virus, and to methods of inducing an immune response or protecting a mammal from infection, using the same.
The invention also includes antibodies to the intact mutant virus and to the mutant viral proteins, and to methods of making and using the same.
Also included in the invention are vectors suitable for delivering a gene to a cell of a human or animal, as well methods of delivery thereof.